RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We recently systematically interrogated RBP dependencies in a number of human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains (RBDs) of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining physiological RNA splicing and AML survival. Genetic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. Fortuitously, it has recently been described that a class of clinically validated anti-cancer sulfonamide compounds (including the drugs indisulam and E7820) mediate RBM39 degradation as their dominant cellular mechanism of action. This occurs via novel interactions between these compounds and the DCAF15 adapter protein of the CUL4/Ddb1 ubiquitin ligase complex with RBM39 as a neo-substrate. Treatment of AML cells with such compounds in vitro and in vivo resulted in similar lethal cellular effects due to perturbations in RNA splicing. The effects of RBM39 loss on splicing resulted in preferential lethality of spliceosomal mutant AML, providing a novel strategy for treatment of AML bearing recurrent mutations in RBPs that regulated splicing. Overall these data identify RBM39 as central to a network of functionally and physically interacting RBPs upregulated in AML over normal hematopoietic precursors and required for AML maintenance. Despite these insights we do not yet understand the basis for the cell- and context-specific roles of RBM39 in malignant versus normal hematopoietic cells. We also do not understand the exact mechanisms by which RBM39 loss results in eradication of AML. We hypothesize that RBM39 is differentially required in malignant versus normal hematopoietic cells, may be differentially required depending on the precise stage of hematopoiesis, and will be required for leukemia initiation as well as maintenance. These hypotheses will be addressed in two Specific Aims. Aim 1 will determine the biological role of RBM39 in normal and malignant hematopoiesis. In this Aim, we will utilize a novel genetic model for genetic deletion of RBM39 in vivo in a cell- and time-specific manner to rigorously dissect the roles of RBM39 in malignant versus normal hematopoietic stem and progenitor cell populations. Aim 2 will identify the mechanistic basis for cell-type and disease-specific roles for RBM39 in normal and malignant hematopoietic cells. In this Aim we will compare the direct RNA binding targets and effects of RBM39 loss on splicing across normal and malignant hematopoietic cells. In addition, we will evaluate a potential novel for RBM39 in transcriptional elongation by the FACT complex, identified by our preliminary studies as an interactor of RBM39.